McKinnon NK , Reeves DC , Akabas MH .

NS030808 NINDS NIH HHS , R01 NS030808 NINDS NIH HHS , T32 GM007288 NIGMS NIH HHS

	Figure 1. The MA-helix structure in the M3–M4 loop region and the portals into the channel’s cytoplasmic vestibule using the 4-à resolution cryo-EM nicotinic ACh receptor structure (Protein Data Bank accession no. 2BG9). Only the MA-helix portion of the M3–M4 loop region structure was resolved. (A) The entire receptor structure is shown, including the largely β-strand extracellular domain, the largely α-helical membrane-spanning domain, and the cytoplasmic MA helix. The first two domains are shown in ribbon cartoon representation, whereas the MA helix is shown in space-filling representation. Each subunit is shown in a different color for illustrative purposes. (B) The structure after removal of the cytoplasmic M3–M4 loop domain. (C) Close-up view from the cytoplasm of a portal into the channel’s cytoplasmic vestibule. The portal region that ions would traverse to enter or leave the vestibule is colored blue. (D) View of the channel’s cytoplasmic vestibule. The MA helices from the front two subunits (orange and tan) have been removed to visualize the cytoplasmic vestibule and the cytoplasmic mouth of the channel. The portals between the MA helices of the remaining three subunits are colored blue. Figure constructed with PyMOL version 1.4.1 and Photoshop version 10.0.1.
	Figure 2. (A) I-V ramp (red) for a WT 5-HT3A receptor. Red curve is the background-subtracted I-V ramp of an oocyte expressing WT 5-HT3A receptor. It is the I-V ramp in the presence of 5-HT (black) minus a ramp in the absence of 5-HT (gray). (B) Normalized I-V ramps from a single oocyte at subsaturating (0.6 µM; black line) and saturating (10 µM; gray line) 5-HT concentrations. Both currents are normalized to the maximum current at −120 mV for the subsaturating 5-HT concentration (−1.8 µA). The maximum current for the saturating 5-HT concentration at −120 mV is −4.5 µA. (C) Tangent line (black) shown on the linear portion from −70 to −30 mV of a WT 5-HT3A I-V curve (red).
	Figure 3. Characterization of the negative slope conductance of the 5-HT3A–glvM3M4 receptor. (A) I-V ramp (red) for a 5-HT3A–glvM3M4 receptor demonstrating the negative slope conductance at the more negative voltages. Red curve is the background-subtracted I-V ramp of an oocyte expressing 5-HT3A–glvM3M4 receptor. It is the I-V ramp in the presence of 5-HT (black) minus a ramp in the absence of 5-HT (gray). All other I-V curves shown are the background-subtracted curves. (B) I-V curves for a 5-HT3A–glvM3M4 receptor in the presence of 1 mM EGTA (blue) and the absence of EGTA (red). (C) I-V curve for the 5-HT3A–SQPAQAA. The negative slope conductance is not present when the Arg residue is removed from the M3–M4 loop. A 5-HT EC30 concentration (400–1,200 nM) was used for the experiments and determined for each oocyte expressing 5-HT3A receptors.
	Figure 4. The effect of substitution of Na+ with organic cations on the I-V relationship of 5-HT3 receptors at saturating concentrations of 5-HT (50 µM). (Top) 5-HT3A WT receptors. (Bottom) Chimeric 5-HT3A–glvM3M4 receptors. Data points at each potential sample are plotted as the mean normalized current ± SEM along with the interpolated fit through the points. The data were normalized for clarity to the current at either +31 or +37 mV, dependent on the exact protocol used.
	Figure 5. Shifts in the reversal potential of I5-HT and changes in relative conductance upon substitution of Na+ with inorganic and organic cations in oocytes expressing either 5-HT3A WT (filled bars) or chimeric 5-HT3A–glvM3M4 (open bars) receptors. (Top) Conductance ratios were calculated taking the slope across the voltage range of −70 to −30 mV for each cation and dividing by the Na+ slope. (Bottom) Reversal potentials derived from the data in Table I were subtracted from those determined in 98 mM Na+ for each subunit. Where a significant difference in ΔVr was observed between the two receptors tested, it is marked with an asterisk (*, P < 0.01; two way ANOVA).
	Figure 6. Comparison of the I-V relationship of the WT 5HT3A and chimeric 5-HT3A–glvM3M4 receptor in cations ≥2.4-à radius. Normalized I-V relationships for WT 5-HT3A in external 98 mM of Na+ solution (gray) or external 98 mM of cation solution (black). (A, C, E, and G) WT 5HT3A and (B, D, F, and H) 5-HT3A–glvM3M4 receptors. (A and B) Li+, (C and D) NH4+, (E and F) methylammonium, and (G and H) ethanolammonium. Both receptors are permeant to cations of <2.4 à . Subsaturating concentrations of 5-HT were used in these experiments.
	Figure 7. Comparison of I-V relationships for WT 5-HT3A and chimeric 5-HT3A–glvM3M4 receptors for cations between 2.6 and 4.5–à radius. Normalized I-V relationship for WT 5-HT3A in external 98 mM of Na+ solution (gray) or external 98 mM of cation solution (black). (A, C, E, and G) WT 5HT3A and (B, D, F, and H) 5-HT3A–glvM3M4 receptors. (A and B) 2-Methylethanolammonium, (C and D) diethanolammonium, (E and F) choline, and (G and H) NMDG. Neither receptor is significantly permeable to cations of >4.5-à radius. The midsized cations (between 2.6 and 4.5 à ) had flat I-V relationships, possibly from the cation blocking the pore or from the cation acting as competitive antagonists at the 5-HT–binding site. Subsaturating concentrations of 5-HT were used in these experiments.
	Figure 8. The relationship between relative permeability and ionic radius in 5-HT3 receptors. Plot of the square root of the relative permeability against ionic radius for the series of cations in Table I, along with linear regression lines for each dataset: solid line, WT; dashed line, 5-HT3A–glvM3M4. Note that two cations share the same calculated radius at 2.6 à , and that the measured relative permeabilities are similar. NMDG, which was impermeant, was excluded from the linear regression analysis. The ammonium results were also excluded because of the large difference between the two channels and the confounding effects of ammonium ion substitution on the oocyte endogenous currents.

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